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. Author manuscript; available in PMC: 2012 Dec 8.
Published in final edited form as: Cancer Lett. 2011 Aug 6;311(2):219–229. doi: 10.1016/j.canlet.2011.07.023

Figure 6. Induction of apoptotic cell death after the second course of the sequential combination treatment.

Figure 6

The pictures are representative microphotographs of conventional, formalin-paraffin sections of the tumors after hematoxylin-eosin staining. Apoptotic cells were identified by morphology based on the characteristic nuclear fragmentation and condensation. Apoptotic tumor cell nuclei were counted among 300–400 tumor cell nuclei on H.E. slides in randomly selected, non-necrotic fields (×400) of tumor and expressed as a percentage. Panel A1 illustrates that MSC did not change the incidence of apoptotic tumor cells, as compared to the control (panel A2). Panel B1 visualizes that the first CPT treatment on day 7 induced several apoptotic tumor cells indicated by arrows, but the combination treatment with MSC (panel B2) did not increase the apoptotic incidence significantly. Panel C1 demonstrates that the second irinotecan treatment on day 14 also induces apoptotic tumor cells labeled by arrows and the combination treatment with MSC (panel C2) significantly increased the apoptosis incidence, as seen on the corresponding bar graphs.

The presence of apoptotic cells were confirmed by Tunel immunohistochemical assay. Representative microphotographs of Tunel immunostaining for apoptotic cells (brown) with nuclear counterstaining with haematoxylin (blue). The figure confirms the presence of apoptotic cells in formalin/paraffin sections in FaDu xenografts taken 14 days after the last treatment. Original magnification is ×400 for all panels. Panel D1 shows buffer control for staining specificity (negative control); in panel D2 shows an untreated FaDu xenograft with sporadic apoptotic tumor cells; in panel D3, MSC did not increase the number of apoptotic cells when compared to untreated controls; in panel D4, irinotecan-treated xenograft increased apoptotic tumor cell induction; and in panel D5, the second course of combination treatment resulted in significant increase of apoptotic cell tumor cells (p<0.05) when compared with irinotecan alone.

The corresponding bar graphs show the quantitation of the apoptotic tumor cell fraction (%). * denotes p < 0.05 when compared to irinotecan alone.